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neck sqaumous cell carcinoma by adenovirus-mediated expression of the Nbs1 protein
Int. J. Radiation Oncology Biol. Phys., Vol. 67, No. 1, pp. 273–278, 2007 Copyright © 2007 Elsevier Inc. Printed in the USA. All rights reserved 0360-3016/07/$–see front matter
doi:10.1016/j.ijrobp.2006.09.019
BIOLOGY CONTRIBUTION
RADIOSENSITIZATION OF HEAD/NECK SQAUMOUS CELL CARCINOMA BY ADENOVIRUS-MEDIATED EXPRESSION OF THE NBS1 PROTEIN JUONG G. RHEE, PH.D.,*‡ DAQING LI, PH.D.,†‡ MOHAN SUNTHARALINGAM, M.D.,*‡ CHUANFA GUO, PH.D.,‡ BERT W. O’MALLEY, JR., M.D.,†‡ AND JAMES P. CARNEY, PH.D.*‡ *The Radiation Oncology Research Laboratory, Department of Radiation Oncology, †Department of Otolaryngology and ‡ The Marlene and Stewart Greenebaum Cancer Center, University of Maryland School of Medicine, Baltimore, MD Purpose: Local failure and toxicity to adjacent critical structures is a significant problem in radiation therapy of cancers of the head and neck. We are developing a gene therapy based method of sensitizing head/neck squamous cell carcinoma (HNSCC) to radiation treatment. As patients with the rare hereditary disorder, Nijmegen breakage syndrome, show radiation sensitivity we hypothesized that tumor-specific disruption of the function of the Nbs1 protein would lead to enhanced cellular sensitivity to ionizing radiation. Experimental Procedures: We constructed two recombinant adenoviruses by cloning the full-length Nbs1 cDNA as well as the C-terminal 300 amino acids of Nbs1 into an adenovirus backbone under the control of a CMV promoter. The resulting adenoviruses were used to infect HNSCC cell line JHU011. These cells were evaluated for expression of the viral based constructs and assayed for clonogenic survival following radiation exposure. Results: Exposure of cells expressing Nbs1-300 to ionizing radiation resulted in a small reduction in survival relative to cells infected with control virus. Surprisingly, expression of full-length Nbs1 protein resulted in markedly enhanced sensitivity to ionizing radiation. Furthermore, the use of a fractionated radiation scheme following virus infection demonstrates that expression of full-length Nbs1 protein results in significant reduction in cell survival. Conclusions: These results provide a proof of principle that disruption of Nbs1 function may provide a means of enhancing the radiosensitivity of head and neck tumors. Additionally, this work highlights the Mre11 complex as an attractive target for development of radiation sensitizers. © 2007 Elsevier Inc. double-strand break, Mre11, Rad50, Nijmegen breakage syndrome.
INTRODUCTION Cancers of the head and neck are a significant clinical challenge in that nearly 15,000 new cases occur yearly. A large number of these cancers are associated with smoking as a risk factor although recently there has been an increase in the incidence of these types of tumors in nonsmokers. Radiation therapy has been the standard treatment for head and neck tumors for a number of years while more recently combined radiation and chemotherapy have become increasingly popular (1). The major therapeutic challenge of head and neck tumors is twofold. First, the vast majority of patients that undergo treatment have local recurrent failure of the tumor and second, the presence of a number of critical normal structures including the carotid artery, the salivary
glands and the optic nerve that make up the anatomy of this region present significant toxicity problems (2). The therapeutic benefit of radiation is generally considered to be due to DNA damage induced cell killing. The critical lesion generated by radiation is the DNA doublestrand break (DSB) and recent advances have led to a greater understanding of the mechanisms of DSB repair. One of the recently recognized critical components in the cellular response to DSBs is the protein complex made up of the Mre11, Rad50 and Nbs1 proteins (herein referred to as the Mre11 complex) (for review see Petrini and Stracker [3]). Mutations in the NBS1 gene that codes for the Nbs1 protein cause the congenital disorder Nijmegen breakage syndrome (NBS) (4, 5) while hypomorphic mutations in the hMRE11 gene lead to the disorder ataxia telangiectasia-like
Reprint requests to: James P. Carney, 655 West Baltimore Street, BRB 6-011, Baltimore, MD 21201. Tel: (410) 706-4276; Fax: (410) 706-6138; E-mail: [email protected] Daqing LI, Ph.D., and Bert W. O’Malley, Jr., M.D., are currently at the Department of Otorhinolaryngology–Head & Neck Surgery, The University of Pennsylvania Health System, Philadelphia, PA. This work was supported by NIH grants RO1CA87851 (to JPC)
and RO1DE014562 (to BWO) as well as funds from the Marlene and Stewart Greenebaum Cancer Center (BWO). Acknowledgments—The authors wish to thank William F. Morgan and Teresa M. Wilson for many helpful discussions as well as the members of the Carney and O’Malley laboratories. Conflict-of-interest: none. Received Feb 4, 2005, and in revised form Sept 8, 2006. Accepted for publication Sept 9, 2006. 273
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disorder (6). Cells derived from these patients are characteristically radiosensitive as well as having defects in the S-phase checkpoint. Furthermore all three proteins are necessary for cellular survival as mouse knockouts of all three genes induce cellular lethalality (7–9). The Mre11 complex is a stable multimeric complex with the Mre11 protein having nuclease activity while the Rad50 protein is ATP-binding and DNA-binding protein (10 –13). The Nbs1 protein does not appear to have any enzymatic activity although it appears to interact with a number of other proteins in the cellular response to DSBs (14). The architecture of the complex is such that Mre11 plays a central role in that it interacts with both Rad50 and Nbs1 while Nbs1 and Rad50 do not directly interact (15). The initial characterization of Nbs1 by two-hybrid interaction analysis revealed that the C-terminal domain of Nbs1 was responsible for the interaction with Mre11 (4). Additionally, recent work has shown that the Nbs1 protein is a substrate for the ATM kinase in the DNA damage response pathway and the N-terminus of the Nbs1 protein interacts with the E2F1 transcription factor at origins of replication (16 –20). The role of the Mre11 complex in the response to DSBs as well as its requirement for cellular survival makes it an attractive target for therapeutic intervention in cancer. Disruption of complex function would be predicted to render cells sensitive to radiation and potentially be directly cytotoxic. To test this hypothesis we have devised recombinant adenoviruses expressing the full-length Nbs1 as well as the C-terminal domain of Nbs1 and assessed the ability of these viruses to increase the radiation sensitivity of HNSCC cells.
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empty viruses (AdGFP) were 1.05 ⫻ 1010, 1.27 ⫻ 1010, and 1.80 ⫻ 1010 plaque forming units per ml (PFU/ml), respectively, as assessed with methods previously published (21).
Western blot and immunoprecipitation JHU011 cells were seeded in 6-well plates (106 cells in 10 mL) and incubated overnight. Cells were exposed to the viruses (AdNbs1-300, Ad-Nbs1-His, or empty virus) in a 0.5 mL medium at a multiplicity of infection (MOI) of 10 for 2 hrs at 37°C. Fresh medium (10 mL) was then added to each well and incubation was continued for 48 hrs before cell harvest. Western blot and immunoprecipitation were carried out as previously described (4).
Cell survival assay JHU011 cells were seeded in 24-well plates (105 cells in 2 mL). Sixteen hours later, the cells were exposed to the viruses (AdNbs1-300, Ad-Nbs1-His, or empty virus) in 0.25 mL of medium at an MOI of 10 for 2 hrs at 37°C. Fresh medium (2 mL) was then added to each well and incubation was continued for 24 hrs before further treatments. One day after the viral infection, cells were trypsinized, plated (102–104 cells/flask) and incubated for 24 hrs before irradiation. Therefore, the time interval between viral infection and radiation exposure was 48 hrs. Following irradiation, duplicate cultures were incubated for 8 –10 days for colony formation. Cultures were fixed with pure ethanol and stained with 1% crystal violet in ethanol, and colonies with more than 50 cells were counted. Surviving fraction was determined by normalizing to the plating efficiency of the untreated control cells. For dose fractionation, the infected cells were irradiated with the 1st, 2nd, and 3rd doses of 2 Gy at 48, 72, and 96 hrs after the viral infections, respectively. Irradiated cells were trypsinized and plated for survival analysis as described above. All irradiations were carried out using a Seifert X-ray unit (Rich. Seifert & Co., Germany) with an average dose rate of 2 Gy/min.
METHODS AND MATERIALS Cell lines The cells used in this study were originally derived from squamous cell carcinomas of the head and neck patients at the Johns Hopkins Hospital, Department of Otolaryngology, and designated as JHU011. Cells were propagated in nude mice (BALB/c nu/nu) and maintained at the University of Maryland tumor bank. The frozen cells were thawed and grown in RPMI 1640 with a 10% fetal bovine serum supplemented with 1% penicillin and streptomycin, and maintained at 37°C in a humidified 5% CO2 incubator. IMR90 fibroblasts and W1799 cells were obtained from ATCC and maintained as previously described (4).
Statistics All survival fractions (SF) were fitted into the linear quadratic model of the form SF⫽exp(-␣D-D2), where D is a radiation dose. The model parameters (␣, ) and their standard errors were determined by the method of least squares to fit general linear models. Multiple comparisons of radiation survival fractions between treated and untreated control cells were also performed using general linear models and the corresponding p-values were presented. All statistical analyses were performed with the SAS/ STAT version 8 software (SAS Institute Inc., Cary, NC).
RESULTS Vector constructions For the construction of adenoviral vectors (Ad-Nbs1-300 and Ad-Nbs1-His), the cDNAs were cloned into the shuttle vector pAdTrack-CMV, linearized with PmeI, and cotransformed into E. coli strain BJ5183 cells with the adenoviral backbone plasmid pAdEasy-1 that contains a green fluorescence protein (GFP). Recombinants were selected for kanamycin resistance, linearized and transfected into 293 cells for viral packaging. The presence of the respective inserts was confirmed by PCR. The control recombinant adenovirus was also produced without the cDNA insertions (empty virus). These adenoviruses were amplified, purified with cesium chloride ultracentrifugation, and stored at ⫺80°C in single-use aliquots. The viral titer of purified Ad-Nbs1-300, Ad-Nbs1His and
To attenuate Mre11 complex function we designed a construct of the Nbs1 protein that contains the C-terminal 300 amino acids (herein referred to as Nbs1–300) (Fig. 1a). The rationale behind this construct is that this domain contains the hMre11 interaction domain and expression of Nbs1–300 would compete with the endogenous Nbs1 for binding to Mre11 thus disturbing the structure of the Mre11 complex. In addition, we created a full-length Nbs1 construct with a C-terminal 6X His tag (Nbs1-His) (Fig. 1a). The cDNAs coding for both Nbs1–300 and Nbs1-His were engineered into a recombinant replication-defective adenovirus where expression of the Nbs1 constructs is driven by
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tein physically interacts with endogenous hMre11 (Fig. 2b). Thus, Nbs1–300 is being integrated into the Mre11 complex. The presence of the 6X His tag at the C-terminus of Nbs1-His facilitates isolation of this recombinant protein by capture with Ni2⫹-agarose beads. Using this approach we carried out micropurification of Nbs1-His using Ni2⫹-agarose beads from extracts of AdNbs1-His infected cells. Western blot of the eluent from the Ni2⫹-Agarose beads indicates that hMre11 physically interacts with Nbs1-His (Fig. 2c). However, comparison of the ratio of Nbs1 to hMre11 in the input crude extract and the Ni2⫹ purified
Fig. 1. Infection of HNSCC with recombinant adenovirus. (A) The domain organization of the Nbs1 protein is shown at the top. The protein contains a Forkhead-associated domain at the N-terminus (diagonal stripe) followed by a breast cancer C-terminal domain (BRCT, cross-hatch). Additionally the protein contains a small domain from amino acids 6665– 695 that is responsible for interacting with hMre11 (fine-hatch). The sites of two ATM phosphorylation sites at Ser 278 and Ser 343 are indicated with stars. Recombinant adenoviruses were generated that contained no cDNA (AdGFP), the full-length Nbs1 protein with a C-terminal 6X His tag (Nbs1-His) and a construct designed to express the C-terminal 300 amino acids of the Nbs1 protein (Nbs1–300). (B) HNSCC cell line JHU011 was infected with empty virus (AdGFP) as well as viruses coding for Nbs1-His and Nbs1–300 and cells incubated 24 hrs. Infection efficiency was measured by examining the infected cells on an inverted fluorescence microscope and comparing the number of GFP positive cells (top) to the total number of cells (bottom). For all of the above viruses there is ⬎95% infection efficiency.
a CMV promoter. A control virus was also produced that lacked any additional protein coding sequence. All viruses contained a second CMV promoter that drives expression of a green fluorescent protein (GFP). When JHU011 cells were infected with adenovirus having AdNbs1–300, AdNbs1His, and AdGFP, nearly all of the cells showed green fluorescence (Fig. 1b). Thus, the recombinant viruses are fully capable of infecting the JHU011 cells. To assess expression of recombinant Nbs1–300 and Nbs1-His from the adenovirus we infected JHU011 cells and subjected crude extracts to Western blot analysis. The expression of Nbs1-His resulted in a slight induction of the steady state level of Nbs1 while the Nbs1–300 was readily detected in extracts from AdNbs1–300 infected cells (Fig. 2a). The rationale of our experimental design predicts that recombinant Nbs1–300 and Nbs1-His should be able to interact with endogenous hMre11. To test this we infected JHU011 cells with AdNbs1–300 virus and 24 hrs post-infection carried out immunoprecipitation with hMre11 antisera. Western blot analysis of the immunopreciptates indicates that the Nbs1–300 pro-
Fig. 2. Assessment of Recombinant Nbs1-His and Nbs1–300 Expression. (A) HNSCC cell line JHU011 was infected with AdGFP, AdNbs1-His and Ad Nbs1–300 and incubated 24 hrs. Crude protein extracts were subjected to SDS-PAGE and Western blot. The data indicate a slight induction of Nbs1-His and expression of the Nbs1–300 at 37 kD specifically in cells infected with AdNbs1– 300. (B) To assess the interaction of Nbs1–300 with endogenous hMre11 extracts from AdGFP infected and AdNbs1–300 infected cells were subjected to immunoprecipitation using hMre11 antiserum. The resulting Western blot was probed with Nbs1- antiserum and demonstrates that the Nbs1–300 is physically associated with endogenous hMre11. (C) To assess the interaction of Nbs1His with endogenous hMre11 extracts from cells infected with AdNbs1-His and Ad Nbs1–300 were used in Ni2⫹-pull-down experiments. Western blot analysis of the pull-down (Ni2⫹) and crude extracts (Crude) from these infected cells was probed with antiserum to Nbs1 and hMre11. The data indicates that the Nbs1His does interact with hMre11 although there appears to be a significant amount of Nbs1-His that is not in a complex with hMre11 as the ratio between the two bands is appreciably different in the pull-down relative to the crude extract.
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Fig. 3. Survival Analysis following expression of Nbs1-His and Nbs1–300. (A) To test the functionality of the recombinant adenoviruses we infected Nijmegen breakage syndrome (NBS) cell line W1799 (Œ)with AdNbs1-His () and AdNbs1–300 (’) and carried out clonogenic survival. As a positive control we also carried out survival analysis with the normal fibroblast cell line IMR90 (). The data indicate that both Nbs1-His and Nbs1–300 are able to complement the radiation sensitivity of the NBS cell line. (B) Head-and-neck squamous cell carcinoma (HNSCC) cell line JHU011 () was infected with AdGFP (Œ), AdNbs1-His ()and AdNbs1–300 (’) and subjected to ionizing radiation 48 hrs post infection. The radiation survival curves indicate that expression of Nbs1–300 results in some increased sensitivity to ionizing radiation at higher doses. However, expression of Nbs1-His results in a significant increase in sensitivity at all doses.
fraction indicates that the amount of hMre11 that interacts with the Nbs1-His is limited. To assess the effect of expression of Nbs1-His and Nbs1– 300 in cells we first confirmed the functionality of the viruses by infecting NBS fibroblast cell line W1799 with AdGFP, AdNbs1–300 and AdNbs1-His and subjected these cell lines to clonogenic survival analysis following ionizing radiation exposure. In addition, we carried out survival analysis of the normal fibroblast cell line IMR90 as a control. Our results indicate that expression of Nbs1–300 and Nbs1-His significantly restore ( p ⬍ 0.01) the levels of survival of the W1799 fibroblast cell line to near wild-type levels (Fig. 3a), in agreement with previous studies (22, 23). This data supports the functionality of our recombinant adenoviruses and confirms previously published observations that not only does the full-length Nbs1-His complement the radiation sensitivity NBS defective cells but the C-terminal domain of Nbs1 similar to our Nbs1–300 construct is also able to complement the radiation sensitivity of NBS cells (22, 23). This is presumably due to the existence of both the hMre11 and ATM interaction domains within this region (24, 25). To examine the effect of expression of Nbs1–300 and Nbs1-His on tumor cells, JHU011 cells were infected
with AdGFP, AdNbs1–300 and AdNbs1-His and 48 hours post-infection subjected to clonogenic survival analysis following ionizing radiation exposure. As shown in Fig. 3b, expression of Nbs1-His induces sensitization at all doses in the dose range of this experiment. Additionally, the expression of Nbs1–300 leads to modest sensitization at higher doses (4 and 5 Gy). The sensitizing effect of Nbs1-His is a surprising result and suggests that cells need a finely tuned level of Nbs1. This may not be unexpected given the function of Nbs1 in a multimeric protein complex with Mre11 and Rad50. Given that standard clinical protocols utilize fractionated exposure of tumors in patients we designed a simple fractionation scheme utilizing the JHU011 cell line in vitro. Following infection with AdGFP, AdNbs1-His and AdNbs1–300, respectively, cells were incubated for 48 hrs and exposed to 2 Gy of X-rays. An aliquot of cells was removed following irradiation and plated for survival analysis. Cultures were further irradiated at 72 and 96 hrs and aliquots similarly removed for survival analysis. The resultant survival changes are shown in Fig. 4. Expression of Nbs1–300 leads to modest sensitization at the second fraction (p ⫽ 0.04) but clear sensitization at the third fraction (p ⬍ 0.001). However, the presence of recombinant Nbs1-His leads to survival that is 10-fold and 30-fold
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Fig. 4. Survival Analysis following fractionated doses of x-rays. HNSCC cell line JHU011 (open) was infected with AdGFP (dots), AdNbs1-His (hatched) and AdNbs1–300 (black) and at 48 hrs post-infection the cultures were irradiated with 2 Gy of X-rays. An aliquot of cells was removed and plated for survival analysis while the rest of the culture was further irradiated at 72 h (2nd fraction) and 96 h (3rd fraction) with 2 Gy per fraction and aliquots similarly analyzed for survival. Surviving fraction is plotted at each dose.
less at the second and third fractions, respectively when compared to cells infected with the AdGFP control virus at the second and third fractions ( p ⬍ 0.001). DISCUSSION In this report we demonstrate that overexpression of both the C-terminal domain of the Nbs1 protein and the entire full-length protein in a head and neck squamous cell carcinoma cell line (HNSCC) leads to sensitization of these cells to ionizing radiation. In addition, we carried out a fractionated exposure scheme of a HNSCC cell line that were infected with the recombinant viruses and we see that the overexpression of the full-length Nbs1 results in an approximately 30-fold sensitization over the course of treating cells with 2 Gy per day for 3 days. These results suggest that the adenovirus-mediated overexpression of Nbs1 may be an
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attractive therapeutic option for sensitizing head and neck squamous cell carcinoma to radiation treatment. Surprisingly, the full-length Nbs1 protein is a significantly better sensitizer. Presently, the explanation for this is unclear although several recent findings lead to a potential hypothesis. While the Mre11 and Rad50 components of the Mre11 complex have clear enzymatic functions no enzymatic activity has been described for Nbs1. However, Nbs1 seems to function as an adaptor molecule that targets the Mre11 complex to double-strand breaks through proteinprotein interaction. Recently this interaction at the site of breaks has been shown to be a dynamic process (26). In addition, the function of Nbs is generally accepted to be that of an adaptor molecule for additional protein-protein interactions. As our data indicates that most of the overexpressed Nbs1-His is not in complex with Mre11 it may be that this free Nbs1-His acts as a competitor for these protein-protein interactions. Nbs1 is required for the S-phase checkpoint following DNA damage and is required for the ATMdependent phosphorylation of SMC1 (27–30). The mechanism of this is only partially understood as the Mre11 complex has recently been shown to be necessary for activation of the ATM kinase following DNA damage (31–33). Thus, the presence of noncomplexed Nbs1 may lead to an attenuation of ATM activation. We are currently pursuing these hypotheses to decipher the mechanism of the radiosensitization induced by adenovirus-mediated expression of Nbs1-His. In looking to the potential clinical applicability of the gene therapy protocol described here, the primary challenges are specificity and efficiency. Specificity is crucial given the ubiquitous nature of the Mre11 complex, as targeting of normal tissue would lead to enhanced sensitivity of this as well as the tumor. Efficiency is critical in that failure to infect a significant number of tumor cells would lead to minimal sensitization of tumors in vivo. Recent developments in selectively replicating adenovirus may be able to answer both of these challenges as these viruses would only replicate in tumor cells providing specificity and the replicating nature would allow for the infection of a large fraction of a given tumor. This is a line of experimentation that we are currently pursuing. Finally, the ubiquitous nature of the Mre11 complex would allow the application of this therapeutic approach to a wide range of tumor types.
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doi:10.1016/j.ijrobp.2006.09.019
BIOLOGY CONTRIBUTION
RADIOSENSITIZATION OF HEAD/NECK SQAUMOUS CELL CARCINOMA BY ADENOVIRUS-MEDIATED EXPRESSION OF THE NBS1 PROTEIN JUONG G. RHEE, PH.D.,*‡ DAQING LI, PH.D.,†‡ MOHAN SUNTHARALINGAM, M.D.,*‡ CHUANFA GUO, PH.D.,‡ BERT W. O’MALLEY, JR., M.D.,†‡ AND JAMES P. CARNEY, PH.D.*‡ *The Radiation Oncology Research Laboratory, Department of Radiation Oncology, †Department of Otolaryngology and ‡ The Marlene and Stewart Greenebaum Cancer Center, University of Maryland School of Medicine, Baltimore, MD Purpose: Local failure and toxicity to adjacent critical structures is a significant problem in radiation therapy of cancers of the head and neck. We are developing a gene therapy based method of sensitizing head/neck squamous cell carcinoma (HNSCC) to radiation treatment. As patients with the rare hereditary disorder, Nijmegen breakage syndrome, show radiation sensitivity we hypothesized that tumor-specific disruption of the function of the Nbs1 protein would lead to enhanced cellular sensitivity to ionizing radiation. Experimental Procedures: We constructed two recombinant adenoviruses by cloning the full-length Nbs1 cDNA as well as the C-terminal 300 amino acids of Nbs1 into an adenovirus backbone under the control of a CMV promoter. The resulting adenoviruses were used to infect HNSCC cell line JHU011. These cells were evaluated for expression of the viral based constructs and assayed for clonogenic survival following radiation exposure. Results: Exposure of cells expressing Nbs1-300 to ionizing radiation resulted in a small reduction in survival relative to cells infected with control virus. Surprisingly, expression of full-length Nbs1 protein resulted in markedly enhanced sensitivity to ionizing radiation. Furthermore, the use of a fractionated radiation scheme following virus infection demonstrates that expression of full-length Nbs1 protein results in significant reduction in cell survival. Conclusions: These results provide a proof of principle that disruption of Nbs1 function may provide a means of enhancing the radiosensitivity of head and neck tumors. Additionally, this work highlights the Mre11 complex as an attractive target for development of radiation sensitizers. © 2007 Elsevier Inc. double-strand break, Mre11, Rad50, Nijmegen breakage syndrome.
INTRODUCTION Cancers of the head and neck are a significant clinical challenge in that nearly 15,000 new cases occur yearly. A large number of these cancers are associated with smoking as a risk factor although recently there has been an increase in the incidence of these types of tumors in nonsmokers. Radiation therapy has been the standard treatment for head and neck tumors for a number of years while more recently combined radiation and chemotherapy have become increasingly popular (1). The major therapeutic challenge of head and neck tumors is twofold. First, the vast majority of patients that undergo treatment have local recurrent failure of the tumor and second, the presence of a number of critical normal structures including the carotid artery, the salivary
glands and the optic nerve that make up the anatomy of this region present significant toxicity problems (2). The therapeutic benefit of radiation is generally considered to be due to DNA damage induced cell killing. The critical lesion generated by radiation is the DNA doublestrand break (DSB) and recent advances have led to a greater understanding of the mechanisms of DSB repair. One of the recently recognized critical components in the cellular response to DSBs is the protein complex made up of the Mre11, Rad50 and Nbs1 proteins (herein referred to as the Mre11 complex) (for review see Petrini and Stracker [3]). Mutations in the NBS1 gene that codes for the Nbs1 protein cause the congenital disorder Nijmegen breakage syndrome (NBS) (4, 5) while hypomorphic mutations in the hMRE11 gene lead to the disorder ataxia telangiectasia-like
Reprint requests to: James P. Carney, 655 West Baltimore Street, BRB 6-011, Baltimore, MD 21201. Tel: (410) 706-4276; Fax: (410) 706-6138; E-mail: [email protected] Daqing LI, Ph.D., and Bert W. O’Malley, Jr., M.D., are currently at the Department of Otorhinolaryngology–Head & Neck Surgery, The University of Pennsylvania Health System, Philadelphia, PA. This work was supported by NIH grants RO1CA87851 (to JPC)
and RO1DE014562 (to BWO) as well as funds from the Marlene and Stewart Greenebaum Cancer Center (BWO). Acknowledgments—The authors wish to thank William F. Morgan and Teresa M. Wilson for many helpful discussions as well as the members of the Carney and O’Malley laboratories. Conflict-of-interest: none. Received Feb 4, 2005, and in revised form Sept 8, 2006. Accepted for publication Sept 9, 2006. 273
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disorder (6). Cells derived from these patients are characteristically radiosensitive as well as having defects in the S-phase checkpoint. Furthermore all three proteins are necessary for cellular survival as mouse knockouts of all three genes induce cellular lethalality (7–9). The Mre11 complex is a stable multimeric complex with the Mre11 protein having nuclease activity while the Rad50 protein is ATP-binding and DNA-binding protein (10 –13). The Nbs1 protein does not appear to have any enzymatic activity although it appears to interact with a number of other proteins in the cellular response to DSBs (14). The architecture of the complex is such that Mre11 plays a central role in that it interacts with both Rad50 and Nbs1 while Nbs1 and Rad50 do not directly interact (15). The initial characterization of Nbs1 by two-hybrid interaction analysis revealed that the C-terminal domain of Nbs1 was responsible for the interaction with Mre11 (4). Additionally, recent work has shown that the Nbs1 protein is a substrate for the ATM kinase in the DNA damage response pathway and the N-terminus of the Nbs1 protein interacts with the E2F1 transcription factor at origins of replication (16 –20). The role of the Mre11 complex in the response to DSBs as well as its requirement for cellular survival makes it an attractive target for therapeutic intervention in cancer. Disruption of complex function would be predicted to render cells sensitive to radiation and potentially be directly cytotoxic. To test this hypothesis we have devised recombinant adenoviruses expressing the full-length Nbs1 as well as the C-terminal domain of Nbs1 and assessed the ability of these viruses to increase the radiation sensitivity of HNSCC cells.
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empty viruses (AdGFP) were 1.05 ⫻ 1010, 1.27 ⫻ 1010, and 1.80 ⫻ 1010 plaque forming units per ml (PFU/ml), respectively, as assessed with methods previously published (21).
Western blot and immunoprecipitation JHU011 cells were seeded in 6-well plates (106 cells in 10 mL) and incubated overnight. Cells were exposed to the viruses (AdNbs1-300, Ad-Nbs1-His, or empty virus) in a 0.5 mL medium at a multiplicity of infection (MOI) of 10 for 2 hrs at 37°C. Fresh medium (10 mL) was then added to each well and incubation was continued for 48 hrs before cell harvest. Western blot and immunoprecipitation were carried out as previously described (4).
Cell survival assay JHU011 cells were seeded in 24-well plates (105 cells in 2 mL). Sixteen hours later, the cells were exposed to the viruses (AdNbs1-300, Ad-Nbs1-His, or empty virus) in 0.25 mL of medium at an MOI of 10 for 2 hrs at 37°C. Fresh medium (2 mL) was then added to each well and incubation was continued for 24 hrs before further treatments. One day after the viral infection, cells were trypsinized, plated (102–104 cells/flask) and incubated for 24 hrs before irradiation. Therefore, the time interval between viral infection and radiation exposure was 48 hrs. Following irradiation, duplicate cultures were incubated for 8 –10 days for colony formation. Cultures were fixed with pure ethanol and stained with 1% crystal violet in ethanol, and colonies with more than 50 cells were counted. Surviving fraction was determined by normalizing to the plating efficiency of the untreated control cells. For dose fractionation, the infected cells were irradiated with the 1st, 2nd, and 3rd doses of 2 Gy at 48, 72, and 96 hrs after the viral infections, respectively. Irradiated cells were trypsinized and plated for survival analysis as described above. All irradiations were carried out using a Seifert X-ray unit (Rich. Seifert & Co., Germany) with an average dose rate of 2 Gy/min.
METHODS AND MATERIALS Cell lines The cells used in this study were originally derived from squamous cell carcinomas of the head and neck patients at the Johns Hopkins Hospital, Department of Otolaryngology, and designated as JHU011. Cells were propagated in nude mice (BALB/c nu/nu) and maintained at the University of Maryland tumor bank. The frozen cells were thawed and grown in RPMI 1640 with a 10% fetal bovine serum supplemented with 1% penicillin and streptomycin, and maintained at 37°C in a humidified 5% CO2 incubator. IMR90 fibroblasts and W1799 cells were obtained from ATCC and maintained as previously described (4).
Statistics All survival fractions (SF) were fitted into the linear quadratic model of the form SF⫽exp(-␣D-D2), where D is a radiation dose. The model parameters (␣, ) and their standard errors were determined by the method of least squares to fit general linear models. Multiple comparisons of radiation survival fractions between treated and untreated control cells were also performed using general linear models and the corresponding p-values were presented. All statistical analyses were performed with the SAS/ STAT version 8 software (SAS Institute Inc., Cary, NC).
RESULTS Vector constructions For the construction of adenoviral vectors (Ad-Nbs1-300 and Ad-Nbs1-His), the cDNAs were cloned into the shuttle vector pAdTrack-CMV, linearized with PmeI, and cotransformed into E. coli strain BJ5183 cells with the adenoviral backbone plasmid pAdEasy-1 that contains a green fluorescence protein (GFP). Recombinants were selected for kanamycin resistance, linearized and transfected into 293 cells for viral packaging. The presence of the respective inserts was confirmed by PCR. The control recombinant adenovirus was also produced without the cDNA insertions (empty virus). These adenoviruses were amplified, purified with cesium chloride ultracentrifugation, and stored at ⫺80°C in single-use aliquots. The viral titer of purified Ad-Nbs1-300, Ad-Nbs1His and
To attenuate Mre11 complex function we designed a construct of the Nbs1 protein that contains the C-terminal 300 amino acids (herein referred to as Nbs1–300) (Fig. 1a). The rationale behind this construct is that this domain contains the hMre11 interaction domain and expression of Nbs1–300 would compete with the endogenous Nbs1 for binding to Mre11 thus disturbing the structure of the Mre11 complex. In addition, we created a full-length Nbs1 construct with a C-terminal 6X His tag (Nbs1-His) (Fig. 1a). The cDNAs coding for both Nbs1–300 and Nbs1-His were engineered into a recombinant replication-defective adenovirus where expression of the Nbs1 constructs is driven by
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tein physically interacts with endogenous hMre11 (Fig. 2b). Thus, Nbs1–300 is being integrated into the Mre11 complex. The presence of the 6X His tag at the C-terminus of Nbs1-His facilitates isolation of this recombinant protein by capture with Ni2⫹-agarose beads. Using this approach we carried out micropurification of Nbs1-His using Ni2⫹-agarose beads from extracts of AdNbs1-His infected cells. Western blot of the eluent from the Ni2⫹-Agarose beads indicates that hMre11 physically interacts with Nbs1-His (Fig. 2c). However, comparison of the ratio of Nbs1 to hMre11 in the input crude extract and the Ni2⫹ purified
Fig. 1. Infection of HNSCC with recombinant adenovirus. (A) The domain organization of the Nbs1 protein is shown at the top. The protein contains a Forkhead-associated domain at the N-terminus (diagonal stripe) followed by a breast cancer C-terminal domain (BRCT, cross-hatch). Additionally the protein contains a small domain from amino acids 6665– 695 that is responsible for interacting with hMre11 (fine-hatch). The sites of two ATM phosphorylation sites at Ser 278 and Ser 343 are indicated with stars. Recombinant adenoviruses were generated that contained no cDNA (AdGFP), the full-length Nbs1 protein with a C-terminal 6X His tag (Nbs1-His) and a construct designed to express the C-terminal 300 amino acids of the Nbs1 protein (Nbs1–300). (B) HNSCC cell line JHU011 was infected with empty virus (AdGFP) as well as viruses coding for Nbs1-His and Nbs1–300 and cells incubated 24 hrs. Infection efficiency was measured by examining the infected cells on an inverted fluorescence microscope and comparing the number of GFP positive cells (top) to the total number of cells (bottom). For all of the above viruses there is ⬎95% infection efficiency.
a CMV promoter. A control virus was also produced that lacked any additional protein coding sequence. All viruses contained a second CMV promoter that drives expression of a green fluorescent protein (GFP). When JHU011 cells were infected with adenovirus having AdNbs1–300, AdNbs1His, and AdGFP, nearly all of the cells showed green fluorescence (Fig. 1b). Thus, the recombinant viruses are fully capable of infecting the JHU011 cells. To assess expression of recombinant Nbs1–300 and Nbs1-His from the adenovirus we infected JHU011 cells and subjected crude extracts to Western blot analysis. The expression of Nbs1-His resulted in a slight induction of the steady state level of Nbs1 while the Nbs1–300 was readily detected in extracts from AdNbs1–300 infected cells (Fig. 2a). The rationale of our experimental design predicts that recombinant Nbs1–300 and Nbs1-His should be able to interact with endogenous hMre11. To test this we infected JHU011 cells with AdNbs1–300 virus and 24 hrs post-infection carried out immunoprecipitation with hMre11 antisera. Western blot analysis of the immunopreciptates indicates that the Nbs1–300 pro-
Fig. 2. Assessment of Recombinant Nbs1-His and Nbs1–300 Expression. (A) HNSCC cell line JHU011 was infected with AdGFP, AdNbs1-His and Ad Nbs1–300 and incubated 24 hrs. Crude protein extracts were subjected to SDS-PAGE and Western blot. The data indicate a slight induction of Nbs1-His and expression of the Nbs1–300 at 37 kD specifically in cells infected with AdNbs1– 300. (B) To assess the interaction of Nbs1–300 with endogenous hMre11 extracts from AdGFP infected and AdNbs1–300 infected cells were subjected to immunoprecipitation using hMre11 antiserum. The resulting Western blot was probed with Nbs1- antiserum and demonstrates that the Nbs1–300 is physically associated with endogenous hMre11. (C) To assess the interaction of Nbs1His with endogenous hMre11 extracts from cells infected with AdNbs1-His and Ad Nbs1–300 were used in Ni2⫹-pull-down experiments. Western blot analysis of the pull-down (Ni2⫹) and crude extracts (Crude) from these infected cells was probed with antiserum to Nbs1 and hMre11. The data indicates that the Nbs1His does interact with hMre11 although there appears to be a significant amount of Nbs1-His that is not in a complex with hMre11 as the ratio between the two bands is appreciably different in the pull-down relative to the crude extract.
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Fig. 3. Survival Analysis following expression of Nbs1-His and Nbs1–300. (A) To test the functionality of the recombinant adenoviruses we infected Nijmegen breakage syndrome (NBS) cell line W1799 (Œ)with AdNbs1-His () and AdNbs1–300 (’) and carried out clonogenic survival. As a positive control we also carried out survival analysis with the normal fibroblast cell line IMR90 (). The data indicate that both Nbs1-His and Nbs1–300 are able to complement the radiation sensitivity of the NBS cell line. (B) Head-and-neck squamous cell carcinoma (HNSCC) cell line JHU011 () was infected with AdGFP (Œ), AdNbs1-His ()and AdNbs1–300 (’) and subjected to ionizing radiation 48 hrs post infection. The radiation survival curves indicate that expression of Nbs1–300 results in some increased sensitivity to ionizing radiation at higher doses. However, expression of Nbs1-His results in a significant increase in sensitivity at all doses.
fraction indicates that the amount of hMre11 that interacts with the Nbs1-His is limited. To assess the effect of expression of Nbs1-His and Nbs1– 300 in cells we first confirmed the functionality of the viruses by infecting NBS fibroblast cell line W1799 with AdGFP, AdNbs1–300 and AdNbs1-His and subjected these cell lines to clonogenic survival analysis following ionizing radiation exposure. In addition, we carried out survival analysis of the normal fibroblast cell line IMR90 as a control. Our results indicate that expression of Nbs1–300 and Nbs1-His significantly restore ( p ⬍ 0.01) the levels of survival of the W1799 fibroblast cell line to near wild-type levels (Fig. 3a), in agreement with previous studies (22, 23). This data supports the functionality of our recombinant adenoviruses and confirms previously published observations that not only does the full-length Nbs1-His complement the radiation sensitivity NBS defective cells but the C-terminal domain of Nbs1 similar to our Nbs1–300 construct is also able to complement the radiation sensitivity of NBS cells (22, 23). This is presumably due to the existence of both the hMre11 and ATM interaction domains within this region (24, 25). To examine the effect of expression of Nbs1–300 and Nbs1-His on tumor cells, JHU011 cells were infected
with AdGFP, AdNbs1–300 and AdNbs1-His and 48 hours post-infection subjected to clonogenic survival analysis following ionizing radiation exposure. As shown in Fig. 3b, expression of Nbs1-His induces sensitization at all doses in the dose range of this experiment. Additionally, the expression of Nbs1–300 leads to modest sensitization at higher doses (4 and 5 Gy). The sensitizing effect of Nbs1-His is a surprising result and suggests that cells need a finely tuned level of Nbs1. This may not be unexpected given the function of Nbs1 in a multimeric protein complex with Mre11 and Rad50. Given that standard clinical protocols utilize fractionated exposure of tumors in patients we designed a simple fractionation scheme utilizing the JHU011 cell line in vitro. Following infection with AdGFP, AdNbs1-His and AdNbs1–300, respectively, cells were incubated for 48 hrs and exposed to 2 Gy of X-rays. An aliquot of cells was removed following irradiation and plated for survival analysis. Cultures were further irradiated at 72 and 96 hrs and aliquots similarly removed for survival analysis. The resultant survival changes are shown in Fig. 4. Expression of Nbs1–300 leads to modest sensitization at the second fraction (p ⫽ 0.04) but clear sensitization at the third fraction (p ⬍ 0.001). However, the presence of recombinant Nbs1-His leads to survival that is 10-fold and 30-fold
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Fig. 4. Survival Analysis following fractionated doses of x-rays. HNSCC cell line JHU011 (open) was infected with AdGFP (dots), AdNbs1-His (hatched) and AdNbs1–300 (black) and at 48 hrs post-infection the cultures were irradiated with 2 Gy of X-rays. An aliquot of cells was removed and plated for survival analysis while the rest of the culture was further irradiated at 72 h (2nd fraction) and 96 h (3rd fraction) with 2 Gy per fraction and aliquots similarly analyzed for survival. Surviving fraction is plotted at each dose.
less at the second and third fractions, respectively when compared to cells infected with the AdGFP control virus at the second and third fractions ( p ⬍ 0.001). DISCUSSION In this report we demonstrate that overexpression of both the C-terminal domain of the Nbs1 protein and the entire full-length protein in a head and neck squamous cell carcinoma cell line (HNSCC) leads to sensitization of these cells to ionizing radiation. In addition, we carried out a fractionated exposure scheme of a HNSCC cell line that were infected with the recombinant viruses and we see that the overexpression of the full-length Nbs1 results in an approximately 30-fold sensitization over the course of treating cells with 2 Gy per day for 3 days. These results suggest that the adenovirus-mediated overexpression of Nbs1 may be an
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attractive therapeutic option for sensitizing head and neck squamous cell carcinoma to radiation treatment. Surprisingly, the full-length Nbs1 protein is a significantly better sensitizer. Presently, the explanation for this is unclear although several recent findings lead to a potential hypothesis. While the Mre11 and Rad50 components of the Mre11 complex have clear enzymatic functions no enzymatic activity has been described for Nbs1. However, Nbs1 seems to function as an adaptor molecule that targets the Mre11 complex to double-strand breaks through proteinprotein interaction. Recently this interaction at the site of breaks has been shown to be a dynamic process (26). In addition, the function of Nbs is generally accepted to be that of an adaptor molecule for additional protein-protein interactions. As our data indicates that most of the overexpressed Nbs1-His is not in complex with Mre11 it may be that this free Nbs1-His acts as a competitor for these protein-protein interactions. Nbs1 is required for the S-phase checkpoint following DNA damage and is required for the ATMdependent phosphorylation of SMC1 (27–30). The mechanism of this is only partially understood as the Mre11 complex has recently been shown to be necessary for activation of the ATM kinase following DNA damage (31–33). Thus, the presence of noncomplexed Nbs1 may lead to an attenuation of ATM activation. We are currently pursuing these hypotheses to decipher the mechanism of the radiosensitization induced by adenovirus-mediated expression of Nbs1-His. In looking to the potential clinical applicability of the gene therapy protocol described here, the primary challenges are specificity and efficiency. Specificity is crucial given the ubiquitous nature of the Mre11 complex, as targeting of normal tissue would lead to enhanced sensitivity of this as well as the tumor. Efficiency is critical in that failure to infect a significant number of tumor cells would lead to minimal sensitization of tumors in vivo. Recent developments in selectively replicating adenovirus may be able to answer both of these challenges as these viruses would only replicate in tumor cells providing specificity and the replicating nature would allow for the infection of a large fraction of a given tumor. This is a line of experimentation that we are currently pursuing. Finally, the ubiquitous nature of the Mre11 complex would allow the application of this therapeutic approach to a wide range of tumor types.
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